In recent years, medicinal research has uncovered a broad spectrum of diseases that can be treated with recombinant proteins. Examples of proteins of human origin are insulin, EPO and G-CSF the dosage forms and kinds of application of which have been described in various European patents. EP 0 430 200 B1 describes the application of human proteins for subcutaneous and intramuscular administration. Medicaments with stabilised human proteins which contain, amongst others, urea or different amino acids, are known from EP 0 306 824 B1. In this patent, EPO and G-CSF are given as examples. EP 0 607 156 B1 describes the production of conserved medicaments with human proteins for infusion or injection purposes.
In general, the term “recombinant” refers to proteins which are prepared using the recombinant DNA technique. These methods comprise cloning of the gene encoding the protein in question, inserting corresponding cDNA or genomic DNA in a suitable vector system and transforming/transfecting said vectors in suitable host organisms (bacteria or eukaryotic cells). If the cloned gene is expressed in the host organism, the corresponding protein can be recovered from the culture supernatant (if the protein expressed is secreted) or from a homogenate of the host organism (if the corresponding protein is expressed in an intracellular manner). Methods for producing recombinant proteins have been described for both animal and plant proteins. An example of the exact procedure for producing a dimeric plant protein is described in EP 0 751 221B1. This patent describes, amongst others, the unprecedented successful cloning of the genes encoding the ML-subunits. Furthermore, in this patent, the use of said dimeric plant proteins produced recombinantly for the preparation of medicaments is described, too.
The use of mistletoe extracts (extracts of Viscum album) as a curative has been known for centuries. Active ingredients called lectins have been identified as effective components of these extracts. These lectins are proteins which recognise very specific carbohydrate structures also in lipid- or protein-bound form and which bind thereto. Mistletoe lectin which has been characterised as ribosome-inactivating class II protein is pharmacologically effective by the interplay of its two subunits. The B-chain of the mistletoe lectin which has sequence motifs with specific carbohydrate binding properties is, in this case, responsible for the transport of the protein to the target cell. In the target cell, the A-subunit then blocks the ribosomal metabolism of the cell due to its enzymatic rRNA-N-glycosidase activity and, in this way, it triggers a programmed cell death (apoptosis) in said cell.
The pharmaceutical preparations which have so far been known from the state of the art generally contain human proteins, humanised proteins, extracts containing plant proteins or proteins isolated from plants. It is decisive for the effectiveness of preparations containing proteins that the biological activity of said proteins is maintained. For rViscumin, it is, for example, the dimeric structure and the activities that are to be attributed to the single chains and the specific pharmacological mode of action of said molecules that are to be maintained. Maintaining these biological activities strongly depends on the pH of the solution containing the proteins (cf. FIG. 1). Furthermore, storage conditions of the preparation in question influence the stability of a drug/medicament.
The mode of action of the mistletoe plant and the extracts obtained therefrom for treating diseases has been described in European patent EP 0 602 686 B1. As explained in this specification, mistletoe extracts have been used for therapeutic purposes for centuries. Since the beginning of this century, mistletoe preparations are used in cancer therapy with varying success (Bocci, 1993; Gabius et al., Gabius & Gabius, 1994; Ganguly & Das, 1994). Hajto et al. (1989, 1990) were able to show that the therapeutic effects are mediated in particular by so-called mistletoe lectins (viscumins, Viscum album agglutinins, VAA). Apart from a cytotoxic effect, nowadays, in particular an (unspecific) immunostimulation is discussed, the positive effects of which are utilised for an accompanying therapy and for aftercare of tumour patients. An increase in the life quality of such patients is possibly mediated by the release of enogenous endorphins (Heiny and Beuth, 1994).
Numerous tests in vitro (Hajto et al., 1990; Mannel et al., 1991; Beuth et al., 1993a) and in vivo (Hajto, 1986; Hajto et al., 1989; Beuth et al., 1991; Beuth et al., 1992), as well as clinical studies (Beuth et al., 1992) prove the increased release of inflammatory cytokines (TNF-α, IL-1, IL-6) mediated by mistletoe lectin as well as an activation of cellular components of the immune system (TH cells, NK cells).
Today a 60 kDa mistletoe lectin protein is considered an active principle of the mistletoe extracts, wherein the mistletoe lectin can be recovered from extracts in a biochemical manner (Franz et al., 1977; Gabius et al., 1992). The ML protein consists of two covalently S—S-coupled subunits, the A-chain of which is responsible for an enzymatic inactivation of ribosomes (Endo et al., 1988) and the B-chain of which is responsible for the carbohydrate binding. The biological activity is correlated with obtaining the lectin activity of the B-chain (Hajto et al., 1990).